Nucleic acid molecule for enhancing antitumor activity of T cells

A cell and nucleic acid technology, used in the preparation of drugs for the treatment of tumors and the field of chimeric antigen receptors, can solve the problems of weakened CART cell proliferation and survival ability, unsustainable number of CART cells, and difficult to carry out.

Pending Publication Date: 2018-01-12
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the basic principles of tumor immunology and tumor microenvironment, it may be difficult for CART cells to overcome the inhibitory effect of the solid tumor microenvironment in vivo, and its effector functions (such as cell killing, cytokine

Method used

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  • Nucleic acid molecule for enhancing antitumor activity of T cells
  • Nucleic acid molecule for enhancing antitumor activity of T cells
  • Nucleic acid molecule for enhancing antitumor activity of T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, construction of CAR plasmid

[0063] 1) Through gene synthesis, respectively synthesize CAR-Mes (control group), CAR-Mes-DAP10, CAR-Mes-41BB, CAR-Mes-DAP10*3, CAR-Mes-DAP10*9, CAR-Mes-41BB- CD28, CAR-Mes-41BB-DAP10, CAR-PSCA (control group), CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3-41BB, CAR-GPC3- DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10 molecules (the schematic diagram of each molecule is shown in figure 1 shown), the C-terminal of the synthetic gene contains a restriction endonuclease Pme1 restriction site and its protective base and the N-terminus contains a restriction endonuclease AsiSI restriction site and its protective base; wherein, "41BB" It means that the molecule contains the intracellular signaling domain 4-1BB commonly used in the prior art.

[0064] For the gene sequence of each molecule synthesized, please refer to SEQ ID NO: 2 (CAR-Mes), SEQ ID NO: 3 (CAR-Mes-DAP10), SEQ ID NO: 4 (CAR-Mes-41BB), SEQ ID ...

Embodiment 2

[0068] Example 2, lentiviral packaging of CAR plasmid

[0069] The CAR plasmid of the present invention prepared in Example 1 and related control plasmids were packaged with lentivirus, and the specific steps were as follows:

[0070] 1) Cultivate 293T cells in a 10cm culture dish, the medium is: DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 × penicillin-streptomycin mixed solution);

[0071] 2) When the 293T cell density in the 150mm culture dish reaches 80-90%, replace the medium: DMEM high glucose medium + 1% FBS + 1% double antibody;

[0072] 3) After replacing the medium and culturing for 2-6 hours, use PEI to separate the LEGO-CAR-EGFP plasmids (LEGO-CAR-Mes-2A-EGFP, LEGO-CAR-Mes-41BB-2A-EGFP, LEGO-CAR-Mes -DAP10-2A-EGFP, LEGO-CAR-Mes-DAP10*3-2A-EGFP, LEGO-CAR-Mes-DAP10*9-2A-EGFP, LEGO-CAR-Mes-41BB-CD28-2A-EGFP, LEGO -CAR-Mes-41BB-DAP10-2A-EGFP, LEGO-CAR-PSCA-2A-EGFP, LEGO-CAR-PSCA-DAP10-2A-EGFP, LEGO-CAR-PSCA-DAP10*3-2A-EGFP, LEGO -C...

Embodiment 3

[0079] Example 3, T cell activation and CAR lentivirus infection

[0080] ① Separation and purification of T cells: The mononuclear cells in the blood were separated by the Ficoll density gradient method, and the red blood cells were removed by lysing with the red blood cell lysate, and then the T cells were sorted by MACS Pan-T magnetic beads;

[0081] ② Dilute the sorted T cells with medium (AIM-V medium + 5% FBS + penicillin 100U / ml + streptomycin 0.1mg / ml to a cell concentration of 2.5×10 6 pcs / ml for use;

[0082] ③ Stimulate T cells by magnetic beads coated with CD2, CD3, and CD28 antibodies (product source: Miltenyi, Germany), that is, the coated magnetic beads and T cells are mixed at a ratio of 1:2, and the final density of T cells should be 5×10 6 A / ml / cm2. After mixing, place at 37°C, 5% CO 2 Incubate the stimulus for 48 hours in the incubator.

[0083] ④Lentiviral transfection of T cells: remove the magnetic beads in the activated T cell-magnetic bead mixture b...

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PUM

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Abstract

The invention relates to a new chimeric antigen receptor (CAR) molecule containing an intracellular segment of DAP 10 protein, nucleic acid for encoding the CAR molecule, cells for expressing the CARmolecule, and use of the CAR molecule in preparation of medicines for treating tumors. The CAR molecule provided by the invention is realized by introducing the intracellular segment of the DAP 10 protein into the traditional second generation of CAR molecule; a downstream signaling pathway of the DAP10 of the T cells is activated after binding of the CAR molecule and a target antigen, so that thekilling effect of the corresponding CAR T cells on solid tumor in vivo and in vitro is improved, and the proliferation and survival abilities of the CAR T cells are enhanced.

Description

technical field [0001] The present invention relates to the technical field of tumor cell immunotherapy, in particular to a chimeric antigen receptor containing the intracellular segment of DAP10 protein, a nucleic acid encoding it and a cell expressing it, and its role in preparing a drug for treating tumors use. Background technique [0002] Chimeric antigen receptor (CAR, Chimeric Antigen Receptors) T cells are T cells that express chimeric receptors on the surface that recognize specific antigens and can transmit signals. CAR T cells play an important role in anti-tumor by expressing chimeric antigen receptor (CAR) molecules. CAR molecules usually include an extracellular segment, a transmembrane region, and an intracellular segment: the extracellular segment is composed of antibody heavy and light chains A single-chain variable region (ScFv) formed by linking variable regions through a peptide segment; the intracellular segment is a chimera of intracellular segments of...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10C12N15/62C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K19/00C12N5/10C12N15/62C12N15/867
Inventor 不公告发明人
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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